Worldwide, invasive fish cause tens of millions of dollars (US$) in ecosystem and fisheries damage annually and are increasingly targeted for chemical or manual removal in streams and lakes. However, piscicides are non-selective and socially/environmentally unacceptable in some locales, while complete population eradication via conventional manual removal methods like netting or electrofishing is unpredictable and often ineffective as a few invasive individuals often survive to re-build large populations (Makhrov et al. 2014). A new method, the YY male or Trojan Y Chromosome (TYC) approach, relies on development of a hatchery broodstock whose progeny (when stocked) incorporate a second Y chromosome into the undesired wild population. Theoretically, if enough YY males are stocked into the target population over time, the population will skew toward all males, eradicating the undesired population upon stocking cessation (Gutierrez and Teem 2006; Them and Gutierrez 2010). Based on the optimistic predictions in the above two theoretical modeling studies, Schill et al. (2016) developed a YY male broodstock for Brook Trout, the first such broodstock specifically developed for field use in eradicating invasive populations. Subsequent pilot field studies indicate that stocked YY males effectively reproduce with wild females and the results are considered proof-of-concept for the TYC approach in Brook Trout (Kennedy et al., in press).
Despite the success in the TYC approach to date, there is a long felt but unsolved need in the art, in that current techniques require at least three generations to develop YY male broodstocks (see Detailed Description below). Even in the case of rapid-maturing species like the Brook Trout, conventional methodologies require 5 years to create suitable numbers for small pilot field trials and 6-7 years for largescale YY male production capability. In later maturing invasive species like Asian carp currently invading the Mississippi River basin (United States) with catastrophic results (see, e.g. Tsehay et al. 2013), it could take 20+ years to create a YY male broodstock using the same conventional approach as that used for Brook Trout. Such long and costly broodstock development times for late-maturing species are unlikely to interest natural resource managers tasked with halting immediate ecosystem damage. The art needs a technique that develops YY male broodstocks in less than three generations, and preferably in a single generation.
Technology described in this disclosure solves the need by providing a TYC broodstock that may preferably be produced in a single generation, thereby substantially reducing the time required to create suitable numbers of YY males to launch an effective interdiction. Although many of the techniques combined to produce this result have existed by themselves for some time, the combination of the techniques as described herein is new, and yields surprising and unexpected results, in that the combination is substantially greater than “the sum of the parts”. The first step in the disclosed process is to use androgenesis to produce YY male fish (rather than the usual XY arrangement). Androgenesis is not new and has been used in limited commercial aquaculture settings and to produce clones for research purposes (Koman and Thorgaard 2007). Likewise, step two in the disclosed process (feminization of YY male fish using estrogenic hormones) has existed for decades for a variety of species (Piferrer 2001). The third step of the disclosed process (identification of an individual fish's sex using genetic methods) is a more recent scientific development but has also been used for a variety of purposes including the development of YY male fish using the much slower three generation approach (Schill et al. 2016). No known prior work has combined the use of these three procedures to produce a YY male broodstock, preferably in a single generation, that may then be genetically diversified with two standard broodstock production techniques in order to be ready for release into the wild.
Indeed, and in sharp contrast, traditional thinking would more likely have suggested gene modification as the technology from which YY male broodstocks could be developed in a single generation. For example, the “daughterless” approach purports to create a genetically modified organism (GMO) via gene splicing whose progeny are genetically programmed to only produce male offspring (Thresher et al. 2004). Although both the daughterless method and the disclosed inventive YY male approach outlined below in the Detailed Description both seek to drive the sex ratio of wild populations to all male, thereby eradicating the undesired population, the two approaches utilize vastly different methodologies. Further, recent study of the “daughterless” approach described initially by Thresher et al. indicates it that, at least as applied to common carp, the methodology still produces “daughters” (Zhang 2016). The methodology is thus suspect and may even be inoperable in addressing species eradication in the field.
Other reported recent work is also worthy of note as background. Jiang et al, appear to have been working contemporaneously and independently in China on similar subject matter to the technology described herein (Jiang et al. 2018). Jiang et al.'s work focuses on androgenesis and to a lesser extent, sex reversal. They report that they considered the Trojan Y chromosome strategy to be one of the most promising methods to eradicate invasive species, but that, in their view, obtaining fertile YY supermales (MYY) and YY physiological female (phenotypic) females (FYY) is a very difficult process. The production of viable sperm-producing MYY common carp is not new (e.g. Bongers et al. 1999, Koman and Thorgaard 2007). However, Jiang et al.'s androgenetic work produced viable androgenetic MYY broodstock, which were then identified by paternity testing and test crossing. Using estrogen exposure to induce sex reversal, Jiang et al. report that they were able to produce feminized males or FXY carp (a result that has also been reported in earlier journals). Some of the Jiang et al. authors appear as inventors on China patent application CN 20171315756, effective filing date May 8, 2017.
Importantly, neither the referenced publication by Jiang et al., nor the referenced China patent application are prior art to this disclosure. However, the apparent contemporaneous independent work by Jiang et al. suggests strongly that the methodologies generally disclosed herein address a long felt and unsolved need (eradication of invasive species) with a nonobvious approach.
References cited in and/or relevant to this Background section:    Bowers, A. B. J., B. Zandieh-Doulabi, C. J. J. Richter, and J. Komen 1999. Viable Androgenetic YY Genotypes of Common Carp Cyprinus carpio L. Journal of Heredity 90(1): 195-198.    Gutierrez, J. B., and J. L. Teem. 2006. A model describing the effect of sex-reversed YY fish in an established wild population: the use of a Trojan Y chromosome to cause extinction of an introduced exotic species. Journal of Theoretical Biology 241:333-341.    Xiang M., X. Wu, K. Chen, H. Luo, W. Yu, S. Jia, Y. Li, Y. Wang. P. Yang, Z. Zhu and W. Hu. 2018. Production of YY Supermale and XY Physiological Female Common Carp for Potential Eradication of this Invasive Species. Journal of The World Aquaculture Society 10.1111/jwas.12492. See also China patent application CN 20171315756, effective filing date May 8, 2017.    Kennedy, P., K. A. Meyer, D. J. Schill, M. R. Campbell, N. Vu, and N. V. Vu. in press. Survival and reproductive success of hatchery YY male Brook Trout stocked in Idaho Streams. Transactions of the American Fisheries Society.    Koman, H. and G. H. Thorgaard 2007. Androgenesis, gynogenesis and the production of clones in fishes: a review. Aquaculture 269:150-170.    Makhrov, A. A., D. P. Karabanov, and Y. V. Koduhova, 2014. Genetic methods for the control of alien species. Russian Journal of Biological Invasions 5:194-202.    Piferrer, F. 2001. Endocrine sex control strategies for the feminization of teleost fish. Aquaculture 197:229-281.    Schill D. J, J. A. Heindel, M. R. Campbell, K. A. Meyer, and E. R. J. M. Mamer, 2016. Production of a. YY Male Brook Trout broodstock for potential eradication of undesired Brook Trout populations, North American Journal of Aquaculture 78:72-83.    Them, L., and J. B. Gutierrez, 2010. A theoretical strategy for eradication of Asian Carps using a Trojan Y Chromosome to shift the sex ratio of the population. American Fisheries Society Symposium 74:1-12.    Thresher, R, Groyne, P., Patil, J. and L. Hinds, 2004. Genetic control of sex ratio in animal populations. United States Patent Application Publication 2004/0073959 A1.    Tsehay, I, Catalano, M., Sass, G. Glover, D. and B Roth, 2013. Prospects for fishery-induced collapse of invasive Asian Carp in the Illinois River. Fisheries 38: 44-454.    Zhang. D. 2016. Transgenic Disruption of Aromatase Using the Daughterless Construct to Alter Sex Ratio in Common Carp, Cyprinus Carpio. M. S. Thesis, Auburn University.